CA2234664A1 - A method for inhibiting attachment of h. influenzae to human cells using phosphorylated recombinant human beta-casein - Google Patents

Abstract

A method is provided for inhibiting the attachment of H. influenzae to human cells, such as pharyngeal cells, by contacting the bacilli with phosphorylated recombinant human .beta.-casein that have at least 3 phosphate groups. A
product containing phosphorylated recombinant human .beta.-casein may be a liquid enteral nutritional product such as infant formula. The enteral nutritional product may be used for the prevention and treatment of otitis media in infants. The phosphorylated recombinant human .beta.-casein may also be administered as a throat spray or used in nose drops or a spray.

Description

-CA 02234664 1998-0~-06 W O 97tl7085 , PCTAUS96/17860 A METHOD FOR INHIBITING Al~ACHMENT OF H. INFLUENZAETO HUMAN CELLS
USING PHOSPHORYLATED RECOMBINANT HUMAN BETA~ASEIN
This application is a continuation-in-part of U.S. Serial No. 08/249,556 filed May 26, 1994.

TFCHNICAL FIELD

This invention relates to a method for inhibiting the attachment of Haemophilus Influenzae (H. influenzae3 bacilli to human nasopharyngeal cells. More 10 particularly, the invention relates to a method of inhibiting the attachment of H.
influenzae to human cells by contacting the bacilli with phosphorylated recombinant human beta casein containing 3 to 5 phosphate groups.

BACKGROUND OF THE INVENTION

It is generally recognized that human milk is the best nutritional source for human infants. Human milk is not only an ideal source of nutrients for the developing infant, but also contains both immunoglobulins and non-immunological factors that protect the infant from infection by various organisms. Human milk is also easily digested by 20 the infant and is less likely to cause allergic reactions than is infant formula based on bovine milk.

Human milk differs from bovine milk as well as the milk of other mammalian species in various ways. Overall protein content and the kinds of protein differ25 between human and bovine milk. Four major bovine caseins have been identified.
Bovine milk contains 2 a-caseins plus ~- and K-casein, but human milk contains only ~- and K-casein. Additionally, the amino acid sequences of human milk protein differ from that of other mammalian milk proteins.

SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 ~ PCTAUS96/17860 Efforts have been made to develop infant milk formula that have some of the advantaç~eous properties of human milk and avoid the disadvantages associated with bovine milk based infant formulas such as allergic reactions and incompletedigestion by the infant. An intuitively desirable method to achieve this is to add to the formula some of the known constituents of human milk, including human milk proleins in their native form. The human caseins, which differ in amino acid se~uence from their bovine and other mammalian counterparts, represent important sllbst~nces which, if added in their native form to infant formula, would serve to enhance the nutritional value of the formula and reduce the inherent disadvantages of non-human milk proteins.

In addition to being a source of amino acids necessary for the synthesis of protelns required for the growth and development of infants, human milk is recognized as containing proteins, including casein, that have other important biological functions. ~-casein is one of the most abundant milk proteins synthesized in the mammary gland. After post-translational modification in the Golgi apparatus, it is excreted as large calcium-dependent aggregates called micelles. ~-casein is not a sin~le entity, but is a heterogeneous group of phosphoproteins secreted duringlactation in response to lactogenic hormones. The primary structure of human ~-casein was determined by Greenberg et al.(Journal of Biological Chemistry 259:5132-5138, 1984). It was shown to be a phosphorylated protein with phosphorylation sites at specific seryl and threonyl residues located near the amino terminus. Comparison of human and bovine ~-caseins showed 47% identity. The se~uence of human 1c-casein was determined by Brignon et al. ~Feder~tion of Furope~n Biolo~ical Societies I etters 188:48-54, 198~). Whereas ~-casein is phosphorylated, lc-casein is glycosylated.

Several biological effects have been ascribed to human milk casein including:
(1) enhancement of calcium absorption; (2) inhibition of angiotensin l-converting SUBSTITUTE SHEET (RULL 26) CA 02234664 1998-0~-06 W O 97117085 , PCTAUS96rl7860 enzyme; ~3) opioid agonism; (4) and immunostimulating and immunomodulating effects.

Human casein consists largely (>80%) of the ~-form with a smaller amount in the lc-form (Greenberg et al., 1984). Native ~-casein is a 25 kDa protein. In human milk, ~-casein molecules show variable degrees of post-translational phosphorylation ranging from zero to five phosphate groups per polypeptide chain (Greenberg et al., 1g84; Hansson et al.. Protein Expression and Purification 4:373-381, 19g3).
Phosphate groups in the native protein are attached to serine and threonine 10 ressdues located near the amino terminus (Greenberg et al., 1984).

Expression of exogenous genes in bacterial cells provides a useful method for producing recombinant eukaryotic proteins. However, bacteria, such as E. coli, are not capable of producing the post-translational modifications required by many 15 eukaryotic proteins as they do not possess the endogenous enzymes necessary to do so. Therefore, eukaryotic protelns produced in E. coll lack the specific post-translational modifications which may occur within the eukaryotic cell, such as glycosylation, phosphorylation, acetylation, or amidation.

Prior to the development of appropriate cloning techniques, the phosphorylation of purified proteins by a kinase was done in vitro using chemical reagents. This process requires the protein substrate and the kinase enzyme to be purified and this is not efficient or cost-effective for commercial purposes. The in vitro process is also inefficient when it is desired to scale-up for commerci~ tion.

2 5 There is, therefore, a need to develop a method for genetically engineering micr~organisms to phosphorylate a protein in vivo.

Canadian Patent Application No. 2,083,~21 to Pawson et al. teaches a method of producing phosphorylated exogenous protein in host cells. The method of Pawson et al. requires two vectors to be introduced into a bacterial cell. One vector SUBSTITUTE SHEEr (RULE 26) CA 02234664 l998-0~-06 W O 97/17085 , PCT~US96/17860 has a nucleotide sequence encoding an exogenous protein that is capable of beingphosphorylated by the catalytic domain of a proteTn kinase. The other vector has a nuc[eotide sequence encoding the protein kinase catalytic domain. Both vectors are introduced into E. coli and production of the exogenous protein and the protein 5 kinase catalytic domain is induced so that the exogenous protein is phosphorylated.
The bacterial cells are then Iysed and the exogenous phospholylated protein is isolated using standard isolation techniques.

CA No. 2,083,521 does not suggest or disclose the method of the instant 10 invention. The present inventors use a single vector expressing both the substrate and the kinase enzyme. The method of Pawson et al. requires the use of two vectors. The expression system disclosed herein results in specific phosphorylation of the exogenous protein as determined by antibody to phosphoserine, while the expression system of Pawson et al. results in nonspecific phosphorylation of both 1~ host proteins and exogenous proteins. This would adversely affect the growth of host bacteria in scale-up efforts for industrial applications. The present invention, unlike that of Pawson et al., provides for high level production of a phosphorylated, recombinant protein suitable for commercial production.

Simcox et al., Strate~ies in molecular biolo~y 7(3):68-69 (1994) constructed two E. collstrains that harbor a tyrosine kinase plasmid. These TK (tyrosine kinase) strains can be used for generating phosphorylated proteins when transformed witha plasmid containing sequences encoding a phosphorylation target domaln or protein. Both E. colistrains carry an inducible tyrosine kinase gene. One strain, TKB1, is useful for expressing genes whose expression is directed by the T7 promoter. The system developed by Simcox et al. differs from the present invention in that it requires two constructs, i.e., a tyrosine kinase-containing plasmid and a plasmid vector containing a gene encoding a protein or domain to be phosphorylated.

SUBSTITUTE SHEET (RULE 26) =
CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 In order to better understand the structure and function of human ,13 -casein and to permit studies of factors that affect regulation of its synthesis and secretion, cDNA for this protein was cloned and sequenced (Lonnerdal et al., Federation of Furopean 13iolo~ical Societies Letters 269:153-156,1990), and human 5 milk ~-casein was produced in Escherichia coli and Saccharomyces cerevisiae (Hansson et al., 1993). Hansson et al. demonstrated that recombinant human ~-casein was expressed in the yeast, S. cerevisiae, using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA). Production levels were estimated to be approximately 10% of the production found in E coli. However, recombinant, ~-10 casein obtained from S. cerevisiae, a eukaryotic cell that has endogenous enzymescapable of phosphorylating proteins, was phosphorylated, but the protein produced by E. coli, a prokaryotic cell that lacks the ability in its native state to phosphorylate, was non-phosphorylated. Subsequently, it was shown that recombinant human casein kinase ll (rhCKII) produced in and purified from E. coli can 15 phosphorylate protein substrates in vitro (Shi et al., Proceedin~ of the National A~demy of Sciences. USA 91:2767-2771, 1~94). One specific embodiment of the present invention uses a nucleotide sequence encoding a recombinant human caseinkinase ll in a single construct with nucleotide sequence encoding ~-casein to transform E. coli and produce phosphorylated ~ -casein. None of the prior art 20 suggests or discloses a single vector containing a promoter followed by a nucleotide sequence encoding a protein followed by a nucleotide sequence encoding a kinase as is disclosed in the present invention.

SUMMARY OF THE INVENTION
There is disclosed herein a method for producing a modified recombinant - protein in a hos~ cell comprising preparing a single vector encoding both an exogenous protein and an enzyme capable of modifying the exogenous protein.
Representative of exogenous proteins capable of being modified through the 30 process of the present invention include but are not limited to human caseins, SUE~STITUTE SHEEr (RULE 263 CA 02234664 1998-0~-06 W O 97/17085 PCT~US96/17860 including ~-casein, cell receptor proteins, fatty acylated proteins including palmitoylated proteins, mammalian muscle proteins, the gag polyproteins of retroviruses, and mammalian proteins targeted by retroviral src kinases.
Transmembrane glycoproteins that acquire covalent palmitate after synthesis 5 include the insulin, ~2-adrenergic and transferrin receptors. Proteins that function as cell surface receptors, tyrosine and serine/threonine kinases, their substrates, a phosphatase, G-proteins, and Ca2+ are known to be fatty acylated. Representativeof enzymes useful in the present invention because of their capacity to transferfunctional groups to specific exogenous proteins in a host cell, include but are not 10 limited to kinases, such as tyrosine kinases or casein kinase, transferases, such as mammalian and yeast palrnitoyl transferases, and kinases coded for by the src gene of retroviruses. Representative of promoters useful in the present invention include inducible promoters such as T7, ~PL, ~PR. and Tac and constitutive promoters such as bla and spa. Representative of host cells capable of being 15 transformed and then expressing the modified proteins, include but are not limited to the bacterial cells E. coli K-12 and E. coli B, Bacill(Js species, Lactobacillus species, and SfreptococclJs species and eukaryotic cells such as yeast cells or mammaliancells.

An exogenous protein is one that originates outside the organism that is prodiucing it. The term is sometimes used in the relevant DNA cloning literature also to refer to the recombinant protein produced by the transformed recipient organism. Altematively, an exogenous protein produced using DNA cloning techniques may be referred to as a recombinant protein. The terms will be used interchangeably herein since the distinction is frequently not made in the literature.
However, in discussing the disclosed invention the word "recombinant" will be used to refer to the protein produced by the transformed organism, and "exogenous" will be used when referring to the native, non-recombinant protein or nucleotide seq~ence encoding the protein.

SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 There is disclosed herein a method for producing a modified recombinant protein in a host cell comprising the steps of preparing a single vector having a promoter sequence, an exogenous protein sequence, and a nucleotide sequence encoding an enzyme capable of modifying the exogenous protein; transforming the 5 host cell with the vector; expressing the vector in the host cell whereby the produced enzyme modifies the produced recombinant protein; and isolating the produced, modified recombinant protein. Also disclosed herein in a more specificembodiment of the invention is a method for producing a phosphorylated recombinant protein in a host cell comprising the steps of preparing a single vector 10 having a promoter sequence followed by a nucleotide sequence encoding an exogenous protein capable of being phosphorylated by a protein kinase, followed by a nucleotide sequence encoding a protein kinase capable of phosphorylating the exogenous protein; transforming the host cell with the vector; expressing the vector in the host cell whereby the produced protein kinase phosphorylates the produced5 recombinant protein; and isolating the phosphorylated protein.

More particularly, the present inventors have developed a novel method for producing a modified recombinant human protein in bacterial expression systems in which the resulting recombinant human protein has utility for the inhibition of 20 attachment of H. influenzae to human cells and in the prevention and treatment of otitis media in human infants Using a combination of two human casein kinase encoding sequences, expressing respectively the alpha and beta subunits of the kinase, they demonstrated the in vivo production of recombinant phosphorylated human ~-casein in E coli The sequence coding for human casein kinase 11 was placed 25 in tandem with the sequence coding for ,~-casein with the result that a significant portion of the recombinant ,~-casein produced in E coli was phosphorylated as inhuman milk The method of the present invention can also be used for in vivo specific glycosylation, amidation, or acetylation of recombinant proteins in transformed host cells or for the transfer of fatty acids to appropriate recombinant protein 30 substrates in transformed host cells.
SUBSTITIJTE SHEET (I~ULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCT~US96/17860 In a specific embodiment of the invention, a nucleotide sequence encoding a human casein kinase ll (hCKII ~a) is co-expressed in a single construct with a nucleotide sequence encoding a human ~-casein in a bacterial expression system to 5 achieve efficient in vivo phosphorylation of the appropriate serine and threonine residues of recombinant human ~-casein. Experiments in which a nucleotide sequence encoding hCKII ,l~a and a nucieotide sequence encoding human ~-casein were co-expressed in E coli using a single inducible expression vector demonstrated the ability of recombinant hCKII to phosphorylate recombinant ~-casein in vivo. This was 10 an unexpected, nonobvious result requiring experimentation and inventiveness. As was demonstrated by negative results obtained in early, control experiments the disclosed invention showed unexpected results. The method of the present invention produces useful and beneficial results which will permit the addition of beneficial human proteins to nutritional and pharmaceutical products.

Phosphorylated ~-casein produced using the method of the invention is demonstrated to have the same bioactivity as native human ,~-casein as shown by its ability to inhibit adhesion of H. influenzae to human pharyngeal cells.

2 0 BRIFF DF-SCRIPTION OF lHE DRAWINGS

Figur,e 1 shows physical maps of expression vectors pS637 and pRJB-6 constructedfor inducible intracellular expression in E. coli. 191 base pairs were removed from pS637 to produce PRJB-6.
Figure 2 shows physical maps of expression vectors pRJB-6 and pRJB-9 anillustr~tes how pRJB-6 was cut and ligated to CKII ~ a to form pRJB-9.

SUBSTITUT~ SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCT~US96/17860 Figure 3 shows physical maps of expression vectors pS637 and pRJB-7 and shows how pS637 was cut and ligated to CKII ~a to form pRJB-7. pRJB-7 has T7 promoters in front of both the ~-casein and casein kinase genes.

5 Figure 4 shows the physical map of expression vector pS750, constructed for inducible expression and to mediate production of intracellularly localized protein in E coli.

Figure 5 shows SDS-PAGE of Met-~-casein produced in E. coli EL21 strains and stained with Coomassie Brilliant Blue using the vectors pS750 and pET-11d-CKII ,3a.
The codon for methionine (Met) was placed in front of the ~-casein encoding sequence in the construction of plasmid pS750 because in E. coli and other bacteria the synthesis of their proteins begins with the amino acid methionine. This enables the ribosome to recognize the starting polnt for growth of a polypeptide chain.
Production of intracellular recombinant ,~ -casein is possible only when Met is inserted before the encoding sequence for the protein to be produced. Lane 1: molecular weight marker (Bio-Rad prestained, relative molecular weights 106, 80, 49.5, 32.5, 27.5, 18.~ kDa); iane 2: non-phosphorylated recombinant ,B-casein; lane 3: 5P-~-casein; lane 4: pS750 induced with IPTG in BL21(DE3); lane 5: pS750/pET-11d-CKlI2 0 ~a induced with IPTG in BL21 (DE3); lane 6: pS750 induced with IPTG in BL21(DE3)pLys;S; lane 7: pS750/pET-11d-CKII Bainduced with IPTG in BL21(DE3)pLysS; lane 8: pS750 induced with IPTG in BL21(DE3)pLysE; lane 9:
pS750/pET-11d-CKII ,~a induced with IPTG in BL21(DE3)pLysE cells; lane 10: native ,B-casein with five attached phosphate groups (5P-~-casein). The arrow indicates the ~-casein band.

Figure 6 shows SDS-PAGE of Met-~-casein produced in E coli BL21 strains stained with Ethyl Stains-AII using the vectors pS750 and pET-11d-CKII 13a Lane 1: native ~-casein with five attached phosphate groups (5P-,~-casein); lane 2: pS750/pET-11d-CKII ~a induced with IPTG in BL21(DE3)pLysE cells; lane 3: pS750 induced with IPTG
SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCTAJS96/17860 in Bi ~1(DE3)pLysE; lane 4: pS7~CI/pET-11d-CKII ,Ba induced with IPTG in ~3L21(DE3)pLysS; lane ~: pS750 induced with IPTG in BL21(DE3)pLysS; lane 6:
pS7~0/pET-11d-CKII ~.a induced with IPTG in BL21(DE3); iane 7: pS750 induced with IPT~3 in BL21(DE3); lane 8: SP-i3-casein; lane 9: non-phosphorylated recombinant B-5 casein; lane 10: molecular weight marker (Bio-Rad prestained, relative molecular weights 106, 80, 49.5, 32.5, 27.5, 18.~ kDa). The arrow indicates the phosphorylated ,~-casein band, which is seen as a green band in the original pho~ographs.

Figure 7 shows SDS-PAGE of ,'~Aet-~-casein produced in E. coliHMS174(DE3)pLysS
stained with Ethyl Stains-AII using the vectors pS750 and pET-lld-CKII. Lane 1:
mol0cular weight marker (Bio Rad prestained); lane 2: pS7~0 uninduced; lane 3:
pS7!50 induced with IPTG; lane 4: pS7~0/pET-11d-CKII ~a uninduced; lane ~:
pS750/pET-1 1d-CKII i3a induced with IPTG; lane 6: pET-1 1d-CKI1 13a uninduced; lane 7:
pET-11d-CKII Ba induced with IPTG; lane 8: native 5P-B-casein; lane 9 recombinant ,'3-casein; lane 10: molecular weight marker (Bio-Rad prestained, relative molecularweights 106, 80, 49.5, 32.5, 27.~, 18.~ kDa). The arrow indicates the phosphorylated ~-casein band, which is seen as a green band in the original pho~ographs.
Figure 8 shows a Westem immunoblot analysis using antibody to human i3-casein.
Lane 1: molecular weight marker (Gibco BRL, relative molecular weights 43.1, 29.2, 18.8, 16.5, 6.4 kDa); lane 2: 50 ng native human i3-casein; lane 3: uninduced HMS174(DE3)pLysS(pRJB-7); lane 4: induced HMS174(DE3)pLysS(pRJB-7); lane 5:
uninduced HMS174(DE3)pLysS(pET-11d-CKII i3a); lane 6: induced HMS174(DE3)pLysS(pET-11d-CKII i3a); lane 7: uninduced HMS174(DE3)pLysS(pRJB-9); lane 8: induced HMS174(DE3)pLysS(pRJB-9).

Figure 9 shows a Western immunoblot anaiysis with antibody to phosphoserine.
Lane 1: low molecular weight marker (Gibco BRL, relative mollecular weights 44, SUBSTlTlJTE SHEET (RIJLE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 28.7, 18.~, 14.7, 5.8, 2.9 k~a); lane 2: 1 llg native human ~-casein; lane 3: 2 ~L9 native human ~-casein; lane 5: induced HMS174(DE3~pLysS(pET-~1d-CKII ~oc; lane 6:
induced HMS174(DE3)pLysS(pRJB-9~; lane 7: induced HMS174(DE3)pLysS(pRJB-7);
lane 8: induced HMSI74(DE3)pLysS(pS637); lane 10: 1 ~9 recombinant human ~-casein; lane 11: 2 119 recombinant human ~-casein.

Figure 10 shows an immunoblot analysis using antibody to human ~-casein. Lane 1:molecular weight marker (Gibco BRL, relative molecular weights 44, 28.9, 18.5, 14.7, ~.8 kDa); lane 2: native human ~-casein; lane 3: induced HMS174(DE3)pLysS(pRJB-1 0 9); lane 4: induced HMS174(DE3)pLysS(pS637); lane 5: induced IlMS174(DE3)pLysS(pET-11d-CKII Ba); lane 6: recombinant human ~-casein.

Figure 11 shows an immunoblot analysis using antibody to phosphoserine. Lane 1:
molecular weight marker (Gibco BRL, relative molecular weights 44, 28.9, 18.5, 14.7, 1 5 5.8, 2.9 kDa); lane 2: 1 ~Lg native human ~-casein; lane 3: 500 ng native human ~-casein; lane 4: induced 1~MS174(DE3)pLysS(pRJB-9); lane 5: induced HMS174(DE3)pLysS(pS637); lane 6: induced HMS174(DE3)pLysS(pET-11d-CKII ~a);
lane 7: 1~1g recombinant human ~-casein; lane 8: 500 ng recombinant human ~-casein.
D FTall Fr) DF.SCRIPTION OF THE INVENTION

There is disclosed herein a method for producing a modified recombinant protein in a host cell. In a more specific embodiment the invention relates to amethod for producing a phosphorylated human protein in a bacterial cell. The method comprises the steps of preparing a single vector having both a nucleotidesequence encoding an exogenous protein that is capable of being phosphorylated by a protein kinase and a nucleotide sequence encoding an appropriate protein kinase, expressing the vector in a host cell whereby the produced kinase phosphorylates the produced exogenous protein, and isolating the phosphorylated recombinant SUBSTI~UTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCTrUS96/17860 protein. The present inventors have made the unexpected discovery that placing the nucleotide sequence encoding the protein to be phosphorylated and the nucleotidesequence encoding the kinase in tandem in a single construct with a promoter results in high level and specific phosphorylation while eliminating the negative 5 features ~soci~ted with multiple vectors such as the need for antibiotic resistance genes to be used as markers. Use of the single construct system facilitates scaling up the procedure for industrial use. It is contemplated that the method of the inver~tion will be useful in any host cell system that is capable of expressing the exogenous protein. Suitable host cells include both prokaryotes such as bacteria10 and eukaryotes such as yeast and animal cells.

In the practice of the present invention the preferred host cell is E. coli Nucleotide sequences encoding ,~-casein, in several different expression formats, were evaluated for expression of recom~inant human, ~-casein in an E. co/istrain.
15 After a series of experiments, it was determined that recombinant human ,~-casein was efficiently phosphorylated when sequences encoding human ~-casein were placed in a single construct with sequences encoding human casein kinase CKII ~
Efficiency of phosphorylation was not compromised when both genes were placed intandem in one plasmid when compared with experimental systems in which 20 sequences encoding the kinase and the ~-casein were placed in two separate vectors.

Materials and Methods The following materials and methods were used in the investigations described in Examples 1 to ~. Additional materials and/or methods are described for individual experiments when required. Materials and methods used in Example 6 are separately described.

SUBSTITUTE Sl IEET (RULE 26) CA 02234664 1998-0~-06 W O 97117085 , PCTAUS96/17860 Pl~rnids Plasmid construct pS637 shown in Figure 1 is identical to pS26, constructed and described in Hansson et al., (1993), which is herein incorporated by reference, except that it encodes an additional amino acid, glutamîne (Gln), at position 19. The original expression vector, pS26, was modified to create pS637 which produces a recombinant ,~-casein protein identical to the most abundant variant found in human populations.

The construct pS637 was prepared for co-expression with the nucleotide sequence encoding casein kinase ll (Shi et al., 1994), which is hereby incorporated by reference, by placing the nucleotide sequence encoding CKli ~a, which codes for two casein kinase subunits, ~ and a, as a cassette, downstream from the nucleotide sequence encoding ,~-casein. A three-cistron tandem expression vector pET-11d-CKlI
~a is a plasmid containing CKII ~a that was generated by Shi et al.(1994). First, pS637 was cut at two sites downstream of the ~-casein encoding sequence and religated. A plasmid, pRJB-6, shown in Figure 1, was isolated which had lost 19~bases between the two cut sites. The kinase CKII ~a was prepared for insertion into pRJB-6. After insertion the resulting construct was designated pRJB-9, which is shown in Figure 2. pRJB-9is a single construct designed to mediate production ofphosphorylated ,~-casein. pS637 was also modified to construct the plasmids pS750 and pRJB-7 which will be described in further detail below.

Host Cells In the specific embodiment of the invention described below the host organism transformed by the described vectors was E. coli. Otherrepresentative organisms that could be used with the method of the invention include Bacillus, ~ Lactobacillus, and Streptococcus species.

SUBSTITUTESHEET(RULE26) CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 Promoter In the specific embodiment of the invention described below the 17 promoter was used. Other representative promoters that could be used with the method of the invention include the inducible promoters ~PL and ~PR and Tac and the 5 constitutive promoters bla and spa.

Construction of Pl~mids for P~.cterial l~xpression: Detailed Methods F~ression vector DS637 Expression vector pS637 differs from pS26, described in Hansson et al.
(1993) as it contains a nucleotide triplet encoding the glutamine (Gln) amino acid residlue at position 19 of the ~-casein encoding sequence. This nucleotide sequence was isolated from a human cDNA variant that is more commonly found in human populations than is the sequence of pS26. Two synthetic oligonucleotides were 1 !i synthesized for polymerase chain reaction (PCR) amplification. The synthetic olig~nucleotides provide convenient restriction sites and incorporated codons for amino acids used preferentially by bacteria. The two oligonucleotides were designated SYM4174 (Seq.lD NO: 1) and SYM4175 (Seq.lD N0: 2) and have the following sequences:

SYM4174 5'- CGCTGCAGCATATGCGTGMACCATCGMTC 3' SYM4~17~; ~'- CGGGATCCTGGTCC I ~G I G I I I AA(; I I I I I (~M~,' I I I ~i I (i I I 1~;1 Al~CGGTGATCGAl-

3' 2~ PCR amplification was performed as described in Ausubel et al., (eds.)~urrent Protocols in Molecular Biolo~y. Vol.2, Supp. 16, 15Ø3-t5.1.17 (1991) and the amplified fragment was digested with Pstl and Avall to generate an 85 bp fragnnent. Plasmid pS21, described in Hansson et al. (1993) was digested with EcoRV and Accl and a 328 bp fragment was isolated by gel electrophoresis. The 30 isolated fragment was purified from the agarose gel by electroelution and digested with Avall. This resulted in a 197 bp Avall/Accl fragment which was isolated. The 85 SUBSTITUTE SHEET (RUI E 26) CA 02234664 1998-0~-06 W O 97/17~8~ , PCTAUS96/17860 bp Psfl/Ava 11 digested PCR-amplified fragrnent and the 197 bp Avall/Accl were ligated into Psfl/Accl digested pS25, a plasmid described in Hansson et al. The resulting plasmid construct as sequenced and designated pS636. A 644 bp Ndel and BamHI restriction fragment was isolated from pS636 and introduced into 5 Ndel/BamHI digested vector pS26, a plasmid described in Hansson et al. The resulting expression vector was designated pS637.

Fypre5sion vector pRJB-9 The pET-11d-CKII ~a plasmid comprising the CKII ~a encoding sequences 10 generated by Shi et al. (1994) was prepared for co-expression with recombinant, ~-casein. First, 191 base pairs (bp) were removed from pS637 by cutting two EcoRI
sites downstream from the ,~-casein encoding sequence and religating pS637. A
plasmid, pRJB-6 (Figure 1), was isolated, which had lost the 191 bp between the two sites and had retained a single EcoRV site located 132 bases away from the 3' 15 end of the ~-casein encoding sequence. The plasmid pET-11d-CKII ~3a, containing the CKII ~a, encoding sequence, was cut with Clal and the site was filled in with Klenow enzyme (Stratagene, CA) to create blunt ends. The filled in, C/al cut CKII encoding sequence was inserted into pRJB-6, downstream from the ~-casein encoding sequence, and the resulting construct was designated pRJB-9 and is shown in Figure 20 2.

Fy~res5jon vector pRJB-7 The construct pS637 was prepared for co-expression of recombinant ~-2 5 asein and the CKII ~a kinase by placing the CKII ~a encoding sequence immediately after the ~-casein encoding sequence. The CKII p~a encoding sequence was placed as a Bglll/Bam I fragment into the BamH I site of pS637 and designated pRJB-7. Thisfragment contained the T7 promoter from its original vector, pET-11D-CKII ~~.
Thus, as shown in Figure 3,pRJB-7 contains two T7 promoters, one before the ~-3 0 casein encoding sequence and one before the CKII ~a encoding sequence.
SUBSTITUTE SHEET (RUEE 26) CA 02234664 1998-0~-06 W O 97/17085 ~ PCTrUS96/17860 Expression vector pS750 To change the selective marker from ampicillin resistance to kanamycin resistance, the plasmid pS637 was digested with Pvul and treated with T4 DNA
polymerase to generate blunt ends. The linearized vector was; isolated and ligated with a Hincll kanamycin resistance genblock (Pharmacia, Uppsala, Sweden). The resulting expression vector was designated pS750 (Figure 4).

10 Fxpression vector for recombinant human casein kinase ll The expression vector pET-11d-CKII ~o~ (Shi et al., 1994) was provided by Dr.
C. Walsh of the Harvard Medical School, Boston, MA.

Expression experiments were carried out as described by Studier et al.
(Methods in Fnzvmoloqv 185:60-89, 1990). Bacteria were grown in Luria Broth (LB
medium) containing 50~1g/ml carbenicillin for pET-1 1d-CKII ,~a the plasmid thatcontains a gene conferring resistance to carbenicillin, and 50 ~lg/ml kanamycin for the wector pS750, a plasmid containing a gene conferring resistance to kanamycin.
20 The medium was supplemented with 30 ~Lg/ml chloramphenicol when the strains containing the pLys plasmids, which confer resistance to chloramphenicol, were usecl. For induction of the T7 expression system, the cultures were grown to a density of approximately OD600=O.5, and then 0.4 mM isopropyl ~-D-thiogalactopyranoside (IPTG) was added. The cells were harvested about 90 25 minutes after induction.

Flec~rophoresis and Detection of Recombinant B-Casein Cells were pelleted by centrifugation and the pellet from 1 ml of culture was 30 dissolved in tO0 ,~LI of sample buffer, which contains Tris, glycerol, SDS, dithiothreotol SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 PCT~US96/17860 (DTT), and bromophenol blue. The proteins were separated by SDS-PAGE as described in Laemmli (Nature 227:680-685, 1970). Gradient gels were cast and runin the discontinuous buffer system in a Protean (Bio-Rad, Richmond, CA) electrophoresis unit. Gels were stained as described in Laemmli. Immunoblotting was 5 performed according to the specifications of the manufacturer (Bio-Rad).

Procedure for isol~tion of modified protein The modified protein can be isolated by any standard procedure known to 10 those skilled in the art. Representative of such standard procedures is the following:
Cells are harvested and ruptured by standard mechanical or chemical procedures. Cells are then suspended in buffer, homogenized and centrifuged and the supernatant is discarded. The resulting insoluble pellet is resuspended and the supernatant is discarded. This results in a washed insoluble pellet that is suspended in 60 mM Tris and 6 M Urea at pH 8.2 and homogenized. ~-casein supernatant I is removed resulting in an insoluble extract that is again suspended in 50 mM Tris and 6 M Urea at pH 8.2 and homogenized. ~-casein supernatant ll is removed and supernatants I and ll are pooled. The remaining insoluble extract is discarded. The pooled supernatants are diluted 1:1 with 50 mM Tris and pH 8.2 and treated with 3 20 M Urea to extract ~-casein. The final ,~-casein solution is obtained by dialyzing the Urea extract of ~-casein against 50 mM ethanolamine and 100 mM NaCI at pH 9.5, centrifuging, and diluting in 50 mM ethanolamine, 100 mM NaCI at pH 9.5 to a protein concentration of 5 mg/ml. The pellet is discarded.

2 5 EXAMPI F.~

Examples 1 and 2 are provided to form a basis for the claimed invention, but are not part of the invention being claimed. The experiments described in Examples 1 and 2 show that production of recombinant ~-casein is not adversely affected 30 when bacteria are co-transformed with two vectors containing respectively a S~IBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 PCT~US96/17860 nucleotide sequence encoding ,~-casein and a nucleotide sequence encoding a casein kinase. They also demonstrate that recombinant phosphorylated ~-casein can be prodl~ced using these two vectors in a bacterial system.

Example 3 demonstrates that the precise structure of the single plasmid was neither obvious nor expected, but that its construction required inventiveness and experimentation. Example 4 describes a system in which a single construct, containing a promoter and both the nucleotide sequence coding for the protein tobe transcribed and phosphorylated and the nucleotide sequence coding for the kinase, was used to transform a bacterial strain. In Example 4, production of recombinant phosphorylated ,~-casein using a single plasmid was demonstrated. A
single construct system for expression of extracellularly localized recombinant phospho~ylated ~-casein that is identical to human native ~-casein is described in Example 5. Examples 4 and 5 are within the scope of the presently claimed invention.
Example 6 shows a comparison of six phosphoforms of native human and recornbinant human ,~-caseins made under the direction of the plasmid of the invenltion in their ability to inhibit adhesion of the bacterium H. influenzae to human pharyngeal cells.

F~rrlple 1: Production of B-casein in E.coli ~: Phosphorylation of intracellularly lot~ d recombir~nt Met-~-casein: BL21(DF3) strains To analyze the ability of recombinant human CKII (rhCKII) to phosphorylate recombinant ~-casein in vivo in a bacterial expression system, experiments were performed in E coli using two inducible expression vecl.ors. The expression vector pS7~0 was transformed alone or in combination with expression vector pET-11d-CKII ,50c into the T7 host strains BL21(DE3), BL21(DE3)pLysS, and BL21(0E3)pLysE.
DE3 is a DNA fragment derived from a lambda phage containing a lacl repressor, alacU\J5 promoter which is inducible by isopropyl ~-D-thiogalactopyranoside (IPTG), and a gene for T7 RNA polymerase. In the presence of the inducer, T7 RNA
SlJBSTITUTE SHEET (RIJLE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 polymerase is produced resulting in transcription of the exogenous genes. Plasmid pLysS confers resistance to chloramphenicol and has little effect on growth rateand production of foreign protein. It contains a T7 Iysozyme that increases stability of plasmids in E coll and permits the cells to be Iysed by freezing and thawing.

Results as seen in Figure 5 indicate that high levels of recombinant human Met-~-casein were produced in E. coli and that the amount produced was not influenced by co-production of recombinant human CKII ~a. After electrophoretic separation of the proteins and phosphate staining, CKII ~oc is seen to have 10 phosphorylated recombinant human Met-~-casein in vivo. This is shown in Figure 6 and demonstrates the ability to produce phosphorylated ~-casein in a bacterial system using two vectors. This example is not within the scope of the claims and is provided to assist the examiner in understanding the inventive nature of the invention described in detail in Example 4.

FY~mple 2: ProductTon of B-casein in E.coli K-1~: Phosphorylation of intracellularly localized recombinant Met-~-casein: HMS174(DE3)strains E. coli K-12 strains I IMS1 74(DE3), HMS1 74(DE3)pl ysS, and HMS
20 174(DE3)pLysE were evaluated as hosts for production of recombinant human Met~-casein and were transformed with pS750. The most efficient production was achieved with HMS174(DE3)pLysS. Co-expression experiments using pS750 and pET-11d-CKII ~a showed strong induction of recombinant human Met-~-casein production, which was independent of the presence of pET-11d-CKII ~a. Phosphate 25 staining (Figure 7) showed efficient phosphorylatian of Met-~-casein when co-produced in vlvo with recombinant human CKII. This example, as was also the casefor Example 1 is not within the scope of the claims, and is also provided to assist the examiner in understanding the inventive nature of the invention described inExample 4. A two plasmid system is inherently less desirable than the single plasmid 30 system of the present invention as each of the plasmids must contain an antibiotic SIJBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCT~US96/17860 marker so that its presence in the host cells can bs monitored during the fermentation process. This necessit~tes the use of two antibiotics in the growthmedium and retards bacterial growth.

5 F~rnple 3: Production of human ~-casein E coliK-1~ Construct pRJB-7 containingboth a ~ ein encoding sequence and CKII ~a encoding sequences: T7 Promoter in front of B-casein encoding sequence. T7 Promoter in front of CKII Boc encoding sequences 1 0 The construct pRJB-7, contain~ng the ,~-casein and the (CKII ~oc a genes each preceded by a T7 promoter, was transformed into E. coli K-12 host HMS174(DE3)LysS. The transformation and induction procedures followed were those of the Novagen pET system manual as described in Example 4.

15 Western Blot An~lysis Separation and transfer, blocking and antibody procedures are described in Exarnple 4. Figure 8 shows an immunoblot in which production of ~-casein by E. col HMS174(DE3)LysS cells containing four different constructs is compared. Lysates 20 from both induced and uninduced cell cultures are analyzed. Cells contain pET-11d-CKII ~o~ (plasmid with CKII ~ and a encoding sequences), pRJB-9 (hybrid construct with both ~-casein and CKII ~a encoding sequences and T7 promoter in front of ,~-casein encoding sequence only), or pRJB-7 (hybrid construct with both ,~-casein and CKII ,~o~ encoding sequences and T7 promoters in front of both ,~-casein and CKII ~a 2~ encoding sequences). Transformation of the bacteria with pRJB-7 resulted in severe reduction of bacterial growth. E coli HMS174(DE3)LysS had approximately twice the doubling time as did the same strain transformed with pRJB-9, the construct with only one T7 promoter. The Western blot shown in Figure 8 shows reduced production of rscombinant ~-casein by induced cells containing pRJB-7 when 3 0 compared with cells containing pRJB-9. This is seen by comparing lane 4 (induced SUBSTITUTE SHEET (RUI E 26) CA 02234664 1998-0~-06 W O 97/1708~ PCT~US96/17860 pRJB-7) with lane 8 (induced pRJB-9). Although both pRJB-7 and pRJB-9 are derived from pS637, only pRJB-9 produced amounts of ~-casein equivalent to the parent construct. The presence of an additional T7 promoter before the CKII genes in the hybrid construct had the effect of both reducing cell growth and consequently 5 reducing recombinant protein production.

Figure 9 shows a Western blot analysis in which the Iysates were developed with phosphoserine antibody to detect phosphorylated protein. Induced E. co/i HMS174(DE3)LysS cells containing pET-1 ld-CKII ~a, pRJB-9 (hybrid construct withone T7 promoter), pRJB-7 (hybrld construct with two T7 promoters), or pS637 (contains ,B-casein encoding sequence but not CKII ,Bo~ encoding sequence) were compared for production of phosphorylated recombinant ~-casein. Phosphorylated ~-casein was produced only in cells containing pRJB9 (lane 6). No phosphorylatedprotein was detected in lane 7, which contains the Iysate of cells containing pRJB-7.

Failure to detect phosphorylated protein in the construct with two T7 promoters indicates that both inventiveness and experimentation were required inorder to develop the single construct system disclosed herein for expressing an appropriately modified recombinant protein in microorganisms. Although the 20 experiment with two T7 promoters in a single construct containing the nucleotide sequence encoding a protein and the nucleotide sequence encoding a kinase gave anegative result, under different experimental conditions the use of more than one promoter sequence should not be excluded. Situations where it would be favorableto use two different promoters remain within the scope of the present invention.
F~r~rnple 4: Production of human B-casein in E. co/iK-12: Construct pRJB-9 containing both B-casein encodin~ sequence and CKII ~a encoding sequences The method of producing recombinant human ~-casein disclosed herein uses a 30 single construct expressing both the information for transferring functional groups SUBSTITUTE SHEET (RULE 26~

CA 02234664 1998-0~-06 W O 97/1708~ , PCTAUS96/17860 to specific sites and the protein to be modified. In a specific embodiment of this invention the transferred functional group is phosphate. The transfer is accomplished by a kinase that is demonstrated to mediate phosphorylation of spec:ific sites on recombinant human ~-casein In vivo. It is demonstrated here that 5 not only can human, ,13-casein be specifically phosphorylated ~n vivo by E coli, but that a single-construct with a promoter located before the sequence encoding ~-casein and having the advantages of a single-construct system can successfully mediate this function.

Tr~nsformation into F coliK-12 HMS174CPE3)pLYsS

The construct pRJB-9, containing the ~-casein and CKII ~ genes, was transformed intoE coliK-12 host HMS174(DE3)LysS. The transformation procedure followed was that of the Novagen pET system manual (4th ed., TB No.~5, June, 1 5 1 g94) .

Induction of F~pression E. coli HMS174(DE3)LysS host cells containing plasmids pRJB-9 (Figure 2), pS637 (Figure 1), or pET-lld-CKII ~c (Shi et al, 1994) were grown at 30~C to a density of OD6oo=o.5-o~6. Culture samples were taken before and 6 hours after adding 1 mM of the inducer IPTG. Cells from two 1 ml aliquots were pelleted by centrifugation in a microcentrifuge. Cells were resuspended in sample loading buffer for ~el electrophoresis after which 500 ~l of the supernatants from each aliquotwere collected. The spent culture medium was concentrated in a Microcon 10 spin filter (Amicon) for 35 minutes at 10,000 x G. The retentate was collected after spinning for 3 minutes at 1,000 x G and an equal amount o~ sample buffer at double concentration was added.

SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/1708C, PCTAUS96/17860 Western Blot Analysis Cell Iysates were separated on SDS-Polyacrylamide pre-cast Gel (Integrated Separations System) with a 10-20% gradient and transferred to an Immobilon-P
membrane (Millipore, Bedford, MA) with a semi-dry blotter. Gels were electroblotted at a constant current (0.8 mA/cm2) for 45 minutes onto Immobilon PVDF filters (Millipore) using a Trans-Blot SD Transfer Cell (BioRad) The transfer buffer contained 48 mM Tris, 39 mM glycine, t.3 mM SDS (sodium dodecyl sulfate) and 20% methanol. Prior to transfer, the fi,ter was soaked first in methanol and then in transfer buffer. For Western blot analysis, the membrane was blocked in 3% bovine serum albumin and 0.2% Tween in TBS (25 mM Tris, 0.154 M NaCI, pH 7.4). Primary antibody to ,~,-casein and alkaline phosphatase goat anti-rabbit antibody, the secondary antibody, were diluted 1:8000 in the blocking buffer. An additional antibody was used to detect phosphoserines. Blocking and antibody reactions weredone at 25-26~C in 2% gelatin containing amplification grade porcine skin (U.S.
Biochemicals) in TBS for 2 hours. The blot was then rinsed with TBS for 30 minutes.
Primary antibody, mouse monoclonal anti-phosphoserine (Sigma) was diluted 1:200 or 1:100 in the 2% gelatin blocker and incubated for two hours. The blot was rinsed twice in TBS for 5 minutes. The secondary antibody, goat anti-mouse alkaline phosphatase (Sigma), was diluted 1:4,000 in the gelatin blocker, incubated for one hour, and rinsed as before in TBS. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate were used as substrate for color development.

Figure 10 shows an immunoblot in which production of ~-casein by E. coli K-12 ~MS174(DE3)LysS cells containing three different constructs is compared. Cells contain pS637 (plasmid with ~-casein encoding sequence), pET-11d-CKII ~a (plasmid with CKII ,~ and a encoding sequences), or pRJB-9 (hybrid construct with both ,~-casein and CKII ~,a encoding sequences). Comparison of lanes 3 and 4 shows that the hybrid construct, pRJB-9, is producing equivalent amounts of E,-casein to pS637, 3 0 from which it was derived and which does not contain the CKII ~a encoding SUBSTITUTE SHEET (RULE 26) . ~, CA 02234664 1998-0~-06 W O 97/1708S PCT~US96/17860 seqllences. Both pRJB-9 and pS637 produced betw~en 400-500 mglL of ,~-casein in this host cell. This sxperiment shows that placing the ~-casein encoding sequence in tandem with the encoding sequence for CKII ,~a does not significantly change production of ~-casein.

Figure 11 shows a Western blot analysis in which the Iysates were developed with phosphoserine antibody to detect phosphorylated protein. Increased quantities of native human ~-casein and non-phosphorylated recombinant ~-casein were testedin addition to the Iysates of Figure 8. No phosphorylation of bacterial proteins is 10 seen in lane 6, which contains the Iysate from the CKII ~a plasmid, showing that phosphorylation is specific. The cell Iysate in lane 4, containing pRJB-9 with the, ,B-casein and CKII ,Ba encoding sequences in tandem, shows a strong band cross-reacting with the antibody. The band of lane 4 has the same molecular weight as nati~e human milk ,~-casein by electrophoretic analysis as seen in lanes 2 and 3.
15 There was no cross-reactivity to recombinant, non-phosphorylated human ~-casein, either purified as in lanes 7 and 8 or as expressed In vlvo by pS637 in lane 5. This experiment demonstrates specific, high-level phosporylation of intact, recombinant human ,~-casein in E. coli K-12 in a bacterial system using a single construct.

2 0 F~ample 5: Production of ~-casein in E.coli K-12: Phosphorylation of extracellularly localized recombinant ~ ein: Construct containin~ E. coli leader sequence.
promoter. ~-casein encoding sequence. pDET-11d-CKII Ba in this example there is disclosed the construction of' a single plasmid that is2~; used to transform E. coli K-12 and mediate production of extracellularly localized phosphorylated ,~-casein. To create a single construct designed for secretion ofphosphorylated protein to the periplasmic space of a bacterial cell, the ~-casein encoding sequence is put into an expression vector containing a leader sequence that directs protein transport to the periplasm. A polymerase chain reaction (PCR) 30 is performed using the clone resulting from these procedures as the target DNA.
SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/17085 , PCT~US96/17860 The foliowing primers synthesized at Midland Certified Reagent Co. (Midland, TX) can be used in the PCR, RO4: 5'-TGT AAA ACG GCC ACT-3' (Seq.lD No: 3) and RO-29: 5'-GGG GAT CCG TAC GCG TGA AA C-3' (Seq.l~ No: 4) The base unclerlined in RO-29 incorporates a single base change to create an Mlul site at the end of the ,~-casein 5 encoding sequence in order to eliminate the bacterial initiation codon, methionine, for protein synthesis. This is done so that the resulting protein will have an amino acid sequence identical to that of human ,~-casein. The PCR fragment is then purified. The 3' end of the encoding sequence, which is not modified, is cut with BamH 1. Thisfragment, containing a 5' blunt end and 3' BamH I end, is cloned in the expression 10 vector pET-26b (Novagen, Madison, Wl), which contains a T7 promoter, and cut at the blunt end with Mscl and with BamH 1. The construct described here contains the T7 promoter, but other promoter sequences could be used. The CK11 ~a encoding sequence is inserted as described above for pRJB-9. Expression is induced and Western blot analysis is performed according to the procedures described in 15 Example 4.

A Western blot is performed to identify a protein, isolated from the periplasmic space of the bacterial cells, that cross-reacts with antibody to phosphoserine and migrates similarly to native ~-casein. This experiment 20 demonst~ates phosphorylation of recombinant human ~-casein encoded by a sequence fused to a heterologous translational start and signal sequence, this sequence being preceded by a promoter sequence, and the sequence to be phosphorylated being located in a plasmid containing a kinase encoding sequence such as CKII ~o~. Production o~ extracellularly localized phosphorylated protein has 25 not been previously disclosed either in a one-vector or a two-vector system.

The advantage of extracellular over intracellular localization ol the produced phosphorylated protein lies in the ease of its purification. The periplasmic space of bacterial cells contains less extraneous matter than the interior of the cell so that SU~STITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 wa~ 97/1708~ PCTAUS96/17860 isolation of the purified protein is expedited. This is particularly advantageous during comlmercial production.

FY~mple 6:Comparison of Anti-Adhesion Bioactivity of N~tive and Recombin~nt 5 Human B -Casein Haemophilus are small, gram-negative bacilli with a lipopolysaccharide-protein cell wall and are obligate parasites present on the mucous membranes of humans and animal species. The surface of many but not all strains of Haemophilus 10 ~nfluenza~ is covered with a polysaccharide capsule. Nonencapsulated, nontypeable ~J. J,rJflJJenzae strains colonize the upper respiratory tract in most individuals within the first few months of life and is the species most commonly associated with several diseases including otitis media and sinusitis. (Murray et al., Medical Microbiolo~y. 2d ed., p.260, 1994). They can also exacerbate chronic bronchitis.1 5 An assay was performed to compare the activity of native human ~-casein with recombinant human ~-casein synthesized in cells contaiining pRJB-9 in inhibiting adherence of H. influenzae to human pharynx cellls. Comparisons were made ~etween proteins phosphorylated with 0 to 5 phosphates.
Cells and b~cterial strains Detroit 562 human pharynx carcinoma cells (DT 562) were obtained from the American Type Culture Collection (Rockville, MD). The H. influenzae nontypeable t~ac~erial strain was obtained from Dr. Lauren Bakaletz at the Ohio State University.
~ell culture DT ~62 cells seeded into 96-well plates (Costar, Cambridge, MA) at a density of 20,000-25,000 cells per well were cultured in Dulbeccos's Modlified Eagle Medium (GIESG0, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) 30 ~Hycione, Logan, UT). Cells were incubated in a humidified atmosphere of 95% air SUBSTITUTE SHEET (RU~E 26~

CA 02234664 1998-0~-06 W O 97/17085 , PCTAUS96/17860 and 5% carbon dioxide at 37~C. Experiments were conducted when cells were at least 90% confluent. Plates containing cells were washed three times with 200 IlL of Hanks Balanced Salt Solution ~HBSS) (GIBC0) to remove serum proteins before the addition of bacteria.

Native hum~n ~-casein ~ -casein isolated from human milk was purchased from Symbicom AB, PØ
Box 1451, S-902 24 Umea, Sweden.

10 Separation of Phos~hoforms Cells were harvested by centrifugation at 7000 x 9 for 10 minutes at 40~C.
Supernatant was removed and the pelleted cells were subjected to the freeze/thawmethod described ir~ Johnson et al. (Bio/Technolo~y. December 12, 1994, pp.1357-1360) to release the recombinant ~-casein. After filtration through a 0.45 ,u 15 membrane, samples containing ~-casein were loaded onto an anion exchange column (Mono Q 10/10, Pharmacia Biotech, Uppsala, Sweden). Various phosphoforms were resolved on a linear gradient of 0 to 0.5 M NaCI in 20 mM ethanolamine, 6 M urea, at pH 9.5 over a period of 50 minutes.

Different phosphoforms of recombinant ~-casein were idlentified by comparison of their elution times with those of purified native human milk ~-casein.

P~adiolabeling of bacteria H. fnfllJenzae were streaked onto chocolate agar plates from frozen aliquots of a low passage number and incubated at 37~C in a humidified atmosphere of 95%
air and 5% carbon dioxide for 18 hours to obtain log phase cultures. Bacteria harvested in phosphate buffered saline (PBS) supplemented with 0.0~% bovine serum albumin (BSA) were centrifuged and resuspended in a volume of PBS/BSA
yielding an optical density of 2.4 at a wave length of 600 nm (OD600). 1111ndium-oxine (111ln) (Amersham, Arlington Heights, IL) was used to radiolabel the bacteria.
SUB~TITIJTE Sl I~ET (RULE 26~

CA 02234664 1998-0~-06 W O 97/17085 PCT~US96/17860 50~L~i of the 1111n solution was added to 2.5 ml of the bacterial suspension andincubated for 20 minutes at 37~C. The radiolabeled bacteria were washed twice with 10 ml HBSS to removed unbound 1~l1n and resusupended in 5 rnl HBSS
supp~lemented with 30 nM HEPES buffer (N-2hydroxyethylpeperazine-N'-2-ethane 5 sulfonic acid). 25 ~LL of the l1~1n labeled bacterial suspension were preincubated with 25 ~L of the test agent in a polypropylene 96-well plate for 15 minutes at 37~C to allow binding of the agent to the bacteria.

~uantitation of adhesion 25 ~LL of the preincubation mixture containing radiolabeled bacteria and either native human or recombinant ,~-casein was pipetted into each well of the assay plate containing DT 562 cells. The assay plate was incubated for 20 minutes at 37~C to allow adhesion of the bacteria to the cell monolayer. Nonadhering bacteria were removed by washing the plate three times with HBSS. The assay was 1~ terminated by the addition of 100 ~lL of 0.05 N sodium hydroxide to disrupt the cell monolayer and the adhering H. influenzae. The contents of each well was placed in a Cobra polypropylene tube and counted on a Cobra gamma counter (Packard, Meriden, CT). Results were calculated by averaging the results of four replicates.
Res~lts are presented as tlhe percent inhibition of bacterial adhesion with native 2~) human or recombinant (pRJB-9 ~-casein at 6 different phosphorylation levels when compared to bacterial attachment in control wells containing no test agent.

Results Anti-adhesion activity is only seen consistently when ,~-casein is 2~ phosphorylated with 3, 4, or ~ phosphate groups. At lower levels of phosphorylation little or no anti-adhesion was observed with either native or recornbinant ~-casein. However, at higher phosphoforms when ~-casein had 3, 4, or 5 phosphates there was essentially no difference between the anti-adhesion bioactivity of native or recombinant (pRJB-9) human ~ -caseins. These results show 30 that the bioactivity of ,~-casein in inhibiting adhesion of H. in~luenzae to human SUBSTITUTE SHEET (RULE 26~

W O 97/17085 PCT~US96/17860 pharyngeal cells depends upon the level of phosphorylation. Unphosphorylated or minimally phosphorylated ,B-casein is ineffective. Attachment of 3, 4, or 5 phosphate groups is required for inhibition of adhesion of H. influenze to human pharyngeal cells. Results also demonstrate that phosphorylated recombinant ~-casein made 5 with the plasmid of the invention is as effective as native human ~-casein in inhibiting adhesion of H. influenzae. These results are summarized in Table 1.

ANTI-ADHESION BIOACTIVITY OF NATIV~ AND
RECOMBINANT (pRJB-9) HUMAN BETA-CASEINS

Native H~ Recombinant H~
Pho~photorm T-~t Adh--ionPho-pho~orm T-~t Adh-~lon Conc-ntr-tlonInhlbltlon Conc-ntr~tlon Inhlbltlon ~mDlml) ~ms~lml) 0P 1 .00 1 5% 0P 0.40 -2%
1P 1.00 0% 1P 0.76 -4%
2P 1.00 -1 1% 2P 0.76 35%
3P 1.00 47% 3P 0.76 43%
4P 1.00 52% 4P 0.76 5~ %
5P 1.00 50% 5P 0.76 48%

H. influenzae has been identified as a c~us~tive factor for otitis media (Murray et al., 1994). Since it has been demonstrated in the experiments described ~ above that recombinant human ~-casein phosphorylated in at least three sites under the direction of the plasmid disclosed herein inhibits adhesion of H. influenzae to human cells, it is concluded that phosphorylated recombinant human ,B-casein, as SUBSTITUTE SHEET (RUEE 26) CA 02234664 1998-0~-06 W O 97~1708~ PCTAUS96/17860 described above, may be used in the prevention and treatment of otitis media in humans, particularly in human infants.

Therapeutic effects may be provided by enterally feeding or ingesting an 5 enteral liquid nutritional product, such as infant formula, comprising a therapeutically effec:tive amount of phosphorylated recombinant human ~-casein with 3 or more phosphate groups. Preferably the recombinant phosphorylated human ~-casein used in the practice of the invention is synthesized under the direction of a plasmidconsisting essentially of a promoter, followed by a nucleotide sequence encoding10 human ~-casein, followed by a nucleotide sequence encoding an enzyme that canphosphorylate human ~-casein. It: is understood that an enteral liquid nutritional product, such as an infant. formula, used in the practice of the invention may further contain at ieast one other protein from a mammalian milk, such as human milk, cow'~ milk, or goat's milk, and/or at least one other protein from a vegetable 15 source, such as soybeans or rice. The attachment of H. influenzae to human oropharyngeal cells may also be inhibited by administering via a nasal passageway, or as a throat spray, a formulation containing a therapeutically effective amount of phos;phorylated recombinant human ~-casein. Such a nasally administered formulation may be in the form of either drops or a spray. Administration of 20 enteral, throat spray and nasal products is believed to be effective because the interaction ol human ,~-casein is believed to occur by direct contact in the nasopharynx rather than after ingestion and digestion of the ,~-casein.

This disclosed methodology will allow commercial-scale production of 25 phosphorylated, recombinant mammalian proteins in microorganisms. The disclosed methodology can be used to produce recombinant exogenous protelns, including butnot limited to, recombinant human ,~-casein, in large quantitles. Phosphorylation of ~-casein in a bioreactor makes possible large-scale synthesis in a ferrnentor ofrecombinant ,~-casein that is equivalent to native human ~-casein. This will facilitate 3~) the production of infant formula containing human ~-casein in its native SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97/1708~ . PCTA~S96/17860 phosphorylated state. The disclosed methodology can also be used for phosphorylation of cell proteins, including receptors which are regulated by phosphorylation and dephosphorylation and thereby act as signals in cell metabolism. The disclosed methodology provides a cost-effective method of phosphorylating peptide receptors and will be useful in the manufacture of pharmaceutical drugs.

The single plasmid system is preferable to a two-plasmid system for industrial production of fermented proteins such as recombinant, phosphorylated 1 û human ,~-casein. Large-scale production of rlecombinant protein without the selective pressure provided by antibiotics in the growth medium results in plasmid loss during the fermentation process since the cells containing the plasmids would have no selective advantage over those that contained only one or no plasmids, but wouldbe burdened by the presence of the plasmids resulting in slower growth. However,15 use of multiple antibiotics to provide the selective pressure necessary to maintain both plasmids in the bacteria during fermentation frequently retards bacterial growth and results in lower yield of the desired recombinant product. Therefore, for industrial purposes the single-plasmid system disclosed herein is greatly preferab'le to previously disclosed two-plasmid systems.
Phosphorylated recombinant human ,B-casein with 3 to ~ phosphate groups can be incorporated into any standard or specialized enteral liquid nutritional product including but not limited to infant formulas containing protein from non-human mammalian milk such as bovine or goat milk or protein from vegetable 25 sources such as soybeans or rice, as well as other beverages consumed by young children. A product incorporating phosphorylated recombinant humari ,~-casein having 3 to 5 phosphate groups has utility for the inhibition of attachment of H.
~nfJ~enzae to human cells and in the treatment and prevention of otitis media inhuman infants.

SUBSTITUTE SHEET (RULE 26) CA 02234664 1998-0~-06 W O 97J17085 PCTrUS96117860 The discovery disclosed herein of a novel method for producing recombinant, phosphorylated human ,~-casein, with characteristics similar or identical to ~hat of nati~,~e human ~-casein, makes fe~ble the addition of this protein to infant formula so as to render it more similar to human milk with consequential benefits to ~; developing infants. The disclosure of a method for producing recombinant, modified hur~an proteins in a bacterial system also makes feasible the addition of the human proteins to other food and pharmaceutical products.

Although specific preferred embodiments of the invention have been described 10 above with reference to the accompanying experiments and drawings, it will beapparent that the invention is not limited to those precise embodiments and thatmaroy modifications and variations coulcl be effected by one skilled in the art without departing from the spirit or scope of the invention as defined in the appended claims SUBSTITUTE SI~EET (RULE 2~) W O 97/17085 , PCTrUS96/17860 SEQUENCE LiSllNG
-( 1 ) GENERALINFORMATION:
i) APPLlCANT:Mukerji, P.
Thurmond, J.
Hansson, L.
Baxter, J.
Hards, R.
Leonard, A.
Anderson, S.
Harvey, L.
( i i ) TITLE OF INVENTION: A Method for Inhibiting Attachment of H. Influenzae to Human Cells Using Phosphorylated Recombinant Human Beta-Casein { i i i ) NUMBER OF SEQUCNCF.~: 4 ( i v ) CO~Ht~i~NDENCEADDRESS
( A ) ADD~iStt: Donald O. Nickey ROSS Products Division Abbott Laboratories ( B ) ~ l ~tt l: 625 Cleveland Avenue ( C ) CITY: Columbus D ) STATE: Ohio ( E ) COL.N~Y: United States of America ( F ) ZIP: 4321 5 ( v ) COMPUTER READABLE FORM.
(A) MEDIUMTYPE: Diskette, 3.5 inch, 1.44 Mb Storage (B ) COMPUTER: IBM Compatible C ) OPERATING SYSTEM: MS-DOS Version 6.21 ( D ) SOFTWARE WordPerfect Version 6.0a v i ) CURRENT APPLICATION DATA:
( A ) APPLICATION NUMBER:
( B ) FILING DATE:
~ C ) CLASSIFICATION:
( v i i ) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/249,556 ~B) FILINGDATE: 26-MAY-1994 ( i x ) TELECOMMUNlCATiON INFORMATiON:
(A) TELEPHONE: (614) 624-7080 ( B ) TELEFAX: (614) 624-3074 ( C ) TELEX: None SUBSTITUTE SHEET (RULE 2~) W O 97/1708~ PCT~US96/]7860 ( 2 ) INFORMATION FOR SEQ ID NO: 1 ( i ) SEQUENCE CHARA~; I L. ~ I ICS:
(A) LENGTH: 31 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 CGCl-GCAGCA TATGCGTGAA ACCATCG4AT C 31 (3) INFORMATION FOR SEQ ID NO: 2 (i) SEt;~UENCE CHARACTERISTICS:
(A) Ll_NGTH: 61 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(xi) SE_QUENCE DESCRIPTION: SEQ ID NO: 2 CGGGATCCTG GI ~ i I TTAAC I I I I I CAA~ i I I I GTATTCG GTGATCGATT C 61

(4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 TGTA,~AACGA CGGCCAGT 18

(5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARAC; I t~l~ I ICS:
(A) LENGTH: 21 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA
SUBSTITUTE SHEEr (RULE 26) (~SEQUENCE DESCRIPTION:SEQID NO:4 GkX~GATC ~ T ACG~TE~44A C 21 SUBSTITUTE SHEET (RULE 20)

Claims (17)

What is claimed is:

1. A method of inhibiting the attachment of H. influenzae to human cells by enterally ingesting a liquid nutritional product comprising a therapeuticallyeffective amount of recombinant phosphorylated human .beta.-casein having 3 to 5phosphate groups.

2. A method of inhibiting the attachment of H. influenzae to human cells in a human infant by enterally feeding to the human infant an infant formula comprising a therapeutically effective amount of recombinant phosphorylated human .beta.-casein having 3 to 5 phosphate groups.

3. A method of treating and preventing otitis media in a human by inhibiting the attachment of H. influenzae to human cells by feeding to the human an enteral nutritional product comprising a therapeutically effective amount of recombinant phosphorylated human .beta.-casein having 3 to 5 phosphate groups.

4. A method of treating and preventing otitis media in a human infant by inhibiting the attachment of H. influenzae to human cells by feeding to the human infant an infant formula comprising a therapeutically effective amount of recombinant phosphorylated human .beta.-casein having 3 to 5 phosphate groups.

5. The method of any of claims 1-4 wherein the recombinant phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.

6. The method of either of claims 1 or 3 wherein the liquid enteral nutritional product further comprises at least one other protein from mammalian milk.

7. The method of either of claims 2 or 4 wherein the infant formula further comprises at least one other protein from mammalian milk.

8. The method of either of claims 1 or 3 wherein the liquid enteral nutritional product further comprises at least one other protein from mammalian milk and the phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.

9. The method of either of claims 2 or 4 wherein the infant formula further comprises at least one other protein from mammalian milk and the phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.

10. The method of either of claims 1 or 3 wherein the liquid nutritional product further comprises at least one vegetable protein.

11. The method of either of claims 2 or 4 wherein the infant formula further comprises at least one vegetable protein.

12. The method of either of claims 1 or 3 wherein the liquid nutritional product comprises at least one vegetable protein and the phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.

13. The method of either of claims 2 or 4 wherein the infant formula comprises at least one vegetable protein and the phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.

14. A method of inhibiting the attachment of H. influenzae to human nasopharyngeal cells by administering via a nasal passageway a formulation containing a therapeutically effective amount of phosphorylated, recombinant human .beta.-casein having 3 to 5 phosphate groups.

15. A method of inhibiting the attachment of H. influenzae to human nasopharyngeal cells by administering a throat spray formulation containing a therapeutically effective amount of phosphorylated, recombinant human .beta.-casein having 3 to 5 phosphate groups.

16. A method of inhibiting the attachment of H. Influenzae to human pharyngeal cells by contacting the H. influenzae with phosphorylated, recombinant human .beta.-casein having at least 3 phosphate groups.

17. The method of any one of claims 14, 15, or 16 wherein the phosphorylated human .beta.-casein is synthesized under the direction of a plasmid consisting essentially of:
a. a promoter;
b. followed by a nucleotide sequence encoding human .beta.-casein; and c. followed by a nucleotide sequence encoding an enzyme that can phosphorylate human .beta.-casein.